The use of .alpha.-amino acids has recently developed to a considerable extent, in particular in the fields of medicine and nutrition, and the preparation of .alpha.-amino acids of the L series in particular is becoming more and more important.
At present, three main methods are known for the preparation of L .alpha.-amino acids. These are as follows:
1. The use of hydrolyzates of natural proteins, the limiting phase of which is the separation and the purification of each amino acid.
2. The fermentative method which, although more advantageous than the previous method, does not cover the production of all the desired .alpha.-amino acids.
3. Chemical synthesis which can produce simply and at a low cost large quantities of .alpha.-amino acids, but which has the disadvantage of producing substantially racemic mixtures.
The advantages and the profitability of this third method, which is certainly the most significant for the future from the economic point of view, are closely associated with the availability of general methods for the resolution of racemic mixtures.
Within this context, the introduction of enzymes into the technology of .alpha.-amino acids is presently a much more advantageous method than the conventional methods of physical or chemical separation. The enzymes which are presently used for this purpose may be exopeptidases, for example leucine amino peptidase (LAP) which, from the amide of a DL .alpha.-amino acid, only hydrolyses the L enantiomer. (R Koelsch, Enzymologia, 42,257(1972)). ##STR2##
Carboxypeptidase has been used by a similar mechanism on the N-chloroacetyl derivatives of some amino acids (N. Grubhofer and L. Schleith, Naturwissenshaften, 40,508(1953)). ##STR3##
In these two examples, the yield of L isomer obviously cannot exceed 50%, since the D isomer is not recovered.
An improvement has been made to this enzymatic technique by coupling the action of the enzyme with a chemical type of racemization process (I. Chibata, T. Tosa, T. Sato and T. Mori, Methods in Enzymology, vol.44, p.746, Academic Press, 1976, and I. Chibata. T. Tosa, T. Sato, T. Mori and Y. Matuo, Proceedings of the IVth International Fermentation Symposium: Fermentation Technology Today, p.383, Society of Fermentation Technology, Japan, 1972).
The best known example concerns the use as enzyme of immobilized amino acylase which, from DL N-acetyl amino acids, exclusively hydrolyses the L isomer.
The L amino acid is then separated from the medium by crystallisation after the solvent has been concentrated.
The remaining D acetyl .alpha.-amino acid is then subjected to a treatment with acetic anhydride in an anhydrous medium which results in the formation of a DL 2-methyl 5-oxazoline. This is then hydrolysed to produce DL N-acetyl amino acid which is re-subjected to the action of amino acylase. By a continuous process, this system consequently leads to the complete recovery of the racemic mixture in the form of the L isomer.
In spite of the considerable progress which has been provided by this preparation method, the very great difference in concept should, however, be underlined in this process between the enzymatic catalysis which is used and the racemization stage of a non-catalytic chemical type which necessitates completely different experimental conditions. In effect, after the enzymatic hydrolysis stage which takes place in an aqueous solution, and after a first concentration which allows the separation of the L .alpha.-amino acid which has been produced, the water has to be completely removed and then replaced by acetic anhydride, a reagent which is consumed stoichiometrically in each racemization step. Then, before returning to the enzymatic step, the acetic acid formed in the medium has to be re-evaporated and replaced by water. Although this process is advantageous, it is very onerous from a technical and economic point of view.